Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

Abstract

dAspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.

Fluconazole impacts the extracellular matrix of fluconazole-susceptible and -resistant Candida albicans and Candida glabrata biofilms

“…The production of eDNA has been associated to hyphal growth [58]. Nevertheless, the present study demonstrated that even in the absence of hyphae (e.g.…”

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity

“…The results of these studies add to an emerging body of evidence that fungal biofilms share structural (32)(33)(34)(35) and functional (26,36,37) similarity with those formed by pathogenic bacteria. The finding that glycoside hydrolases can display activity against the exopolysaccharides and biofilms of both fungi and bacteria provides evidence that these similarities could potentially be exploited for the development of therapeutics active against both organisms.…”

“…Similarly, hydrolysis of cationic Pel exopolysaccharide by PelA h enhances the activity of the polycationic antibacterial colistin (22). Interestingly, degradation of biofilm-associated extracellular DNA (eDNA) has been reported to enhance A. fumigatus susceptibility to caspofungin and amphotericin B, although the effects on posaconazole and voriconazole susceptibility were not reported in the study (26). Recent work has suggested that Pel anchors eDNA within P. aeruginosa biofilms through charge-charge interactions (17).…”

“…The Pel polysaccharide enhances resistance to several antibiotics including aminoglycosides and colistin (22,24,25). Because biofilm formation by A. fumigatus is associated with increased resistance to a number of antifungal agents (26)(27)(28), we hypothesized that GAG may have an analogous function to Pel in enhancing resistance to antifungal agents. To test this hypothesis, we investigated whether Sph3 h or PelA h could potentiate the activity of commonly used antifungal drugs.…”

Biofilm Exopolysaccharides of Pathogenic Fungi: Lessons from Bacteria

“…Therapy with recombinant DNase reduces C. albicans biofilm biomass and enhances the activity of amphotericin B, but not fluconazole, in vitro (76,77). Similarly, recombinant DNase was found to disrupt A. fumigatus biofilms and to enhance the activity of several antifungal agents in vitro (78). No studies in animal models evaluating the effects of DNase on antifungal susceptibility have been reported to date.…”